Protein-biotin interactions

The three-dimensional structures of several biotin-binding proteins are now known, giving insights into the molecular architecture of the binding sites for biotin. In combination with biochemical and computational approaches, these structural insights provide the basis for our present understanding of biotin—protein interactions which, in some cases, give rise to spectacular binding constants.     

Chemical approaches toward understanding base excision DNA repair

Despite the importance of DNA repair in protecting the genome, the molecular basis for damage recognition and repair remains poorly understood. In the base excision repair pathway (BER), DNA glycosylases recognize and excise damaged bases from DNA. This review focuses on the recent development of chemical approaches that have been applied to the study of BER enzymes. Several distinctive classes of noncleavable substrate analogs that form stable complexes with DNA glycosylases have recently been designed and synthesized. These analogs have been used for biochemical and structural analyses of protein—DNA complexes involving DNA glycosylases, and for the isolation of a novel DNA glycosylase. An approach to trap covalently a DNA glycosylase-intermediate complex has also been used to elucidate the mechanism of DNA glycosylases.     

Progress towards synthetic enzymes for phosphoester hydrolysis

Synthesis of artificial enzymes for catalyzing phosphoester hydrolysis has been attracting interest for a long time. The remarkable discovery that lanthanide ions catalyze the hydrolysis of DNA and RNA spurred the trend. Currently, progress is being made, mainly in the preparation of homogeneous catalysts, the promotion of catalytic activity by using acid/base cooperation within catalysts, the detailed understanding of the reaction mechanisms involved, and the design of artificial enzymes expressing high specificity and catalytic turn-over.     

Intracellular detection assays for high-throughput screening

New optical assay methods promise to accelerate the use of living cells in screens for drug discovery. Most of these methods employ either fluorescent or luminescent read-outs and allow cell-based assays for most targets, including receptors, ion channels and intracellular enzymes. Furthermore, genetically encoded probes offer the possibility of custom-engineered biosensors for intracellular biochemistry, specifically localized targets, and protein—protein interactions.     

Aminoacyl-tRNA synthetases

Crystallographic studies of a number of aminoacyl-tRNA synthetases and their complexes with ATP, amino acid and cognate tRNA are leading to an increasingly detailed picture of how these sophisticated enzymes function. Within the two distinct structural classes of ten synthetases, many common features are apparent, although evolution has led to many interesting idiosyncrasies in certain enzymes. Recent advances, specially concerning class II enzymes, have increased out knowledge of both the role of electrophiles in the mechanism of amino acid activation and cross-subunit tRNA recognition and help solve the evolutionary puzzles that have emerged from the extension of the aminoacyl-tRNA synthetase database to include Archae     

Photoprocesses

The development of supramolecular chemistry has allowed the construction of structurally organized and functionally integrated chemical systems capable of elaborating the energy and information input of photons to perform complex functions. Model systems capable of mimicking the two fundamental steps of photosynthesis, namely light harvesting and photoinduced charge separation, have recently been developed.     

Active sites of transition-metal enzymes with a focus on nickel

Since 1995, crystal structures have been determined for many transition-metal enzymes, in particular those containing the rarely used transition metals vanadium, molybdenum, tungsten, manganese, cobalt and nickel. Accordingly, our understanding of how an enzyme uses the unique properties of a specific transition metal has been substantially increased in the past few years. The different functions of nickel in catalysis are highlighted by describing the active sites of six nickel enzymes — methyl-coenzyme M reductase, urease, hydrogenase, superoxide dismutase, carbon monoxide dehydrogenase and acetyl-coenzyme A synthase.     

Novel cofactor derivatives and cofactor-based models

In 1997 and the first half of 1998, numerous publications appeared reporting studies of cofactors and their analogues in classical model systems and in enzyme-catalyzed reactions directed at understanding the enzymatic reactions of their natural cofactors. Model systems based on flavins have provided new insights into enzymatic modulation of the flavin reduction potential, and enzymatic reactions of coenzyme A analogues and derivatives have been employed in several studies of coenzyme A utilizing enzymes. Coenzyme B₁₂ analogues have been utilized as alternate cofactors for B₁₂-utilizing enzymes, while pyrroloquinoline quinone esters and analogues have been employed in model studies of the reactions of quinoprotein-catalyzed reactions.     

Structural and mechanistic studies of enolase

The high-resolutionstructure of yeast enolase cocrystallized with its equilibrium mixture of substrate and product reveals the stereochemistry of substrate/product binding and therefore the groups responsible for acid/base catalysis and stabilization of the enolate intermediate. Expression and characterization of site-specific mutant forms of the enzyme have confirmed the roles of amino acid side chains in the catalysis of the first and second steps of the reaction. Coordination of both required magnesium ions to the carboxylate of the substrate/product indicates a role for these cations in stabilization of the intermediate.     

Biosynthetic systems for nonribosomal peptide antibiotic assembly

Modular peptide synthetases, which act as the protein templates for the synthesis of a large number of peptide antibiotics and siderophores, hold great potential for the development of novel compounds. Recently, significant progress has been made towards understanding their molecular architecture and substrate specificity. The first crystal structure of a peptide synthetase has been solved, and the enzymes responsible for post-translational modification of peptide synthetases have recently been discovered. These will allow addressing important yet poorly understood mechanistic aspects.     

Emerging mechanisms of eukaryotic DNA replication initiation

Recent research has focused on proteins important for early steps in replication in eukaryotes, and particularly on Cdc6/Cdc18, the MCMs, and Cdc45. Although it is still unclear exactly what role these proteins play, it is possible that they are analogous to initiation proteins in prokaryotes. One specific model is that MCMs form a hexameric helicase at replication forks, and Cdc6/Cdc18 acts as a ‘clamp-loader’ required to lock the MCMs around DNA. The MCMs appear to be the target of Cdc7-Dbf4 kinase acting at individual replication origins. Finally, Cdc45 interacts with MCMs and may shed light on how cyclin-dependent kinases activate DNA replication.     

Phenylalanine and tyrosine catabolism in some cheese coryneform bacteria

Abstract 23 cheese coryneform bacteria (14 orange, 3 white, and 6 yellow-pigmented) were examined for five enzymes of two branch-point steps in the catabolic pathways of l -phenylalanine and l -tyrosine. Orange cheese coryneforms ( 'Brevibacterium linens' ) catabolized th amino acids by transamination and the benzene ring was cleaved by 3, 4-dihydroxyphenylacetate-2, 3-dioxygenase. Both enzymes appear to be inducible. Yellow and white strains possessed non-inducible low activity of aminotransferase and lacked completely benzene ring cleavage enzymes.     

A role for the cerebellum in the control of limb movement velocity

Accepting, rejecting or modifying the many different theories of the cerebellum's role in the control of movement requires an understanding of the signals encoded in the discharge of cerebellar neurons and how those signals are transformed by the cerebellar circuitry. Particularly challenging is understanding the sensory and motor signals carried by the two types of action potentials generated by cerebellar Purkinje cells, the simple spikes and complex spikes. Advances have been made in understanding this signal processing in the context of voluntary arm movements. Recent evidence suggests that mossy fiber afferents to the cerebellar cortex are a source of kinematic signals, providing information about movement direction and speed. In turn, the simple spike discharge of Purkinje cells integrates this mossy fiber information to generate a movement velocity signal. Complex spikes may signal errors in movement velocity. It is proposed that the cerebellum uses the signals carried by the simple and complex spike discharges to control movement velocity for both step and tracking arm movements.     

Enzyme models: design and selection

There are two approaches to the discovery of enzyme mimics, that is identifying molecules that are able to bind substrate(s) and then catalyze reactions. The first approach, often inspired by enzymes themselves, utilises chemical knowledge and experience to design the catalyst. The other approach is to create a library and select the best host of a transition state analogue of the required reaction.     

Insights into the mechanisms of homologous recombination from the structure of RuvA

The recent structure determination of RuvA has provided the first insights into the structural basis for its interaction with Holliday junction DNA. Multiple copies of a helix-hairpin-helix motif which line the four grooves between the monomers in the tetrameric structure are thought to be involved in the interaction of the protein with its DNA target. This suggests that the four arms of the junction are held by RuvA in a fourfold symmetric arrangement and has fuelled ideas on the way in which components of the Ruv complex combine to catalyse the process of homologous recombination     

Heterocyst formation in Anabaena

Heterocystous cyanobacteria grow as multicellular organisms with a distinct one-dimensional developmental pattern of single nitrogen-fixing heterocysts separated by approximately ten vegetative cells. Several genes have been identified that are required for heterocyst development and pattern formation. A key regulator, HetR, has been recently shown to be aserine-type protease.     

Chain elongation in the isoprenoid biosynthetic pathway

Isoprenyl diphosphate synthases catalyze addition of allylic diphosphate primers to the isoprene unit in isopentenyl diphosphate to produce polyisoprenoid diphosphates with well defined chain lengths. Phylogenetic correlations suggest that the synthases which catalyze formation of isoprenoid diphosphates with (E) double bonds have evolved from a common ancestor. X-ray crystallographic studies of farnesyl diphosphate synthase in conjunction with site-directed mutagenesis have provided important new information about the residues involved in binding and catalysis and the source of chain length selectivity for the enzymes that catalyze chain elongation.     

Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control

Cyclins and cyclin-dependent kinases (Cdks) are universal regulators of cell cycle progression in eukaryotic cells. Cdk activity is controlled by phosphorylation at three conserved sites, and many of the enzymes that act on these sites have now been identified. Although the biochemistry of CdK phosphorylation is relatively well understood, the regulatory roles of such phosphorylation are, in many cases, obscure. Recent studies have uncovered new and unexpected potential roles, and prompted re-examination of previously assumed roles, of Cdk phosphorylation.